scholarly journals Quantifying Green Fluorescent Protein Diffusion inEscherichia coliby Using Continuous Photobleaching with Evanescent Illumination

2009 ◽  
Vol 113 (14) ◽  
pp. 4837-4845 ◽  
Author(s):  
Kristin M. Slade ◽  
Bridgett L. Steele ◽  
Gary J. Pielak ◽  
Nancy L. Thompson
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Protein Diffusion ◽  
Green Fluorescent

scholarly journals Effects of Recombinant Protein Expression on Green Fluorescent Protein Diffusion inEscherichia coli

Biochemistry ◽  
2009 ◽  
Vol 48 (23) ◽  
pp. 5083-5089 ◽  
Author(s):  
Kristin M. Slade ◽  
Rachael Baker ◽  
Michael Chua ◽  
Nancy L. Thompson ◽  
Gary J. Pielak
Keyword(s):  
Green Fluorescent Protein ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Fluorescent Protein ◽  
Recombinant Protein Expression ◽  
Inescherichia Coli ◽  
Protein Diffusion ◽  
Green Fluorescent

A Minimalist Model of Protein Diffusion and Interactions: The Green Fluorescent Protein within the Cytoplasm

2013 ◽  
Vol 46 (20) ◽  
pp. 8311-8322 ◽  
Author(s):  
Fabio Trovato ◽  
Riccardo Nifosì ◽  
Armida Di Fenza ◽  
Valentina Tozzini
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Protein Diffusion ◽  
Green Fluorescent

scholarly journals Impact of signaling microcompartment geometry on GPCR dynamics in live retinal photoreceptors

2012 ◽  
Vol 140 (3) ◽  
pp. 249-266 ◽  
Author(s):  
Mehdi Najafi ◽  
Mohammad Haeri ◽  
Barry E. Knox ◽  
William E. Schiesser ◽  
Peter D. Calvert
Keyword(s):  
Green Fluorescent Protein ◽  
Molecular Diffusion ◽  
Fluorescent Protein ◽  
Lateral Diffusion ◽  
Live Cell ◽  
Confocal Imaging ◽  
Slow Phase ◽  
Protein Diffusion ◽  
Green Fluorescent ◽  
The Impact

G protein–coupled receptor (GPCR) cascades rely on membrane protein diffusion for signaling and are generally found in spatially constrained subcellular microcompartments. How the geometry of these microcompartments impacts cascade activities, however, is not understood, primarily because of the inability of current live cell–imaging technologies to resolve these small structures. Here, we examine the dynamics of the GPCR rhodopsin within discrete signaling microcompartments of live photoreceptors using a novel high resolution approach. Rhodopsin fused to green fluorescent protein variants, either enhanced green fluorescent protein (EGFP) or the photoactivatable PAGFP (Rho-E/PAGFP), was expressed transgenically in Xenopus laevis rod photoreceptors, and the geometries of light signaling microcompartments formed by lamellar disc membranes and their incisure clefts were resolved by confocal imaging. Multiphoton fluorescence relaxation after photoconversion experiments were then performed with a Ti–sapphire laser focused to the diffraction limit, which produced small sub–cubic micrometer volumes of photoconverted molecules within the discrete microcompartments. A model of molecular diffusion was developed that allows the geometry of the particular compartment being examined to be specified. This was used to interpret the experimental results. Using this unique approach, we showed that rhodopsin mobility across the disc surface was highly heterogeneous. The overall relaxation of Rho-PAGFP fluorescence photoactivated within a microcompartment was biphasic, with a fast phase lasting several seconds and a slow phase of variable duration that required up to several minutes to reach equilibrium. Local Rho-EGFP diffusion within defined compartments was monotonic, however, with an effective lateral diffusion coefficient Dlat = 0.130 ± 0.012 µm2s−1. Comparison of rhodopsin-PAGFP relaxation time courses with model predictions revealed that microcompartment geometry alone may explain both fast local rhodopsin diffusion and its slow equilibration across the greater disc membrane. Our approach has for the first time allowed direct examination of GPCR dynamics within a live cell signaling microcompartment and a quantitative assessment of the impact of compartment geometry on GPCR activity.

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